CN103966112A - Hansenula polymorpha recombination strain and application thereof in biosynthesis of gentiopicroside - Google Patents
Hansenula polymorpha recombination strain and application thereof in biosynthesis of gentiopicroside Download PDFInfo
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- CN103966112A CN103966112A CN201410174666.9A CN201410174666A CN103966112A CN 103966112 A CN103966112 A CN 103966112A CN 201410174666 A CN201410174666 A CN 201410174666A CN 103966112 A CN103966112 A CN 103966112A
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- gentiopicrin
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- DUAGQYUORDTXOR-WULZUDSJSA-N Gentiopicrin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1[C@@H](C=C)C=2C(C(=O)OCC=2)=CO1 DUAGQYUORDTXOR-WULZUDSJSA-N 0.000 title claims abstract description 58
- DUAGQYUORDTXOR-GPQRQXLASA-N Gentiopicrin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](C=C)C2=CCOC(=O)C2=CO1 DUAGQYUORDTXOR-GPQRQXLASA-N 0.000 title claims abstract description 58
- 241000320412 Ogataea angusta Species 0.000 title claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 title abstract description 5
- 238000005215 recombination Methods 0.000 title abstract 5
- 230000006798 recombination Effects 0.000 title abstract 5
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims description 54
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 35
- 239000007788 liquid Substances 0.000 claims description 35
- 230000003570 biosynthesizing effect Effects 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 241001071804 Gentianaceae Species 0.000 abstract description 5
- 238000005468 ion implantation Methods 0.000 abstract description 4
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- 238000011161 development Methods 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
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- 241000235342 Saccharomycetes Species 0.000 abstract 3
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
- 241000218671 Ephedra Species 0.000 description 1
- 239000012029 Fehling's reagent Substances 0.000 description 1
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- 240000008917 Glycyrrhiza uralensis Species 0.000 description 1
- 235000000554 Glycyrrhiza uralensis Nutrition 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
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- 235000013617 genetically modified food Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a hansenula polymorpha recombination strain and application thereof in the biosynthesis of gentiopicroside. A recombination saccharomycete used for producing gentiopicroside is obtained by a method for conversion of low energy ion implantation mediated gentiana macrophylla genome total DNA in the saccharomycete, and is named as hansenula polymorpha of which the preservation number is CGMCC.No.8998. The hansenula polymorpha recombination strain and the application realize the technical breakthrough in the aspect of using the recombination saccharomycete to be subjected to biological fermentation to produce gentiopicroside, solves the source shortage of gentiopicroside, protects the gentianaceae plant resources of our country, and provides a new way for sustainable development of the gentianaceae plant resources.
Description
Technical field
The invention belongs to by genetic transformation and obtain the transgenic yeast recombinant bacterium field of producing gentiopicrin, be specifically related to utilize the total DNA of ion implantation mediation bark of ash genetic transformation obtains in yeast multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in gentiopicrin biosynthesizing thereof.
Background technology
Gentiopicrin (Gentiopicroside), another name gentiopicroside, molecular formula is C
16h
20o
9, molecular weight is 356.11, for driffractive ring cyclenes terpene glycosides compound, belongs to sesquiterpenoids material, is the main medicinal ingredients of medicinal plant bark of ash (Gentiana macrophylla).Research in recent years shows, gentiopicrin has the effects such as analgesia, anti-inflammatory, antibacterial, antiviral, anti-oxidant, hepatic cholagogic, stomach invigorating antiulcer agent, as as first class national new drug injection Qinlongkusu, for the freeze-dried powder of Radix Gentianae Macrophyllae extract gentiopicrin, for icteric viral hepatitie, treat.
Gentiopicrin multi-source is in Gentianaceae per nnial herb bark of ash.Bark of ash is Gentianaceae, Gentiana per nnial herb, is one of important Chinese medicinal materials of China.Bark of ash is grown in high altitude localities, and growth cycle is long, is subject to seasonal variation impact.Because bark of ash has very high pharmaceutical use, transition is excavated and is made Wild Gentiana macrophylla resource occur serious scarcity, adds the low problem of bark of ash artificial culture survival rate, causes the supply of gentiopicrin to be difficult to meet clinical demand in recent years.Therefore, in the urgent need to seeking and expanding gentiopicrin source new drugs by way of to meet growing to it clinically demand.
At present, the utilization Agrobacterium rhizogenes such as Tiwari induce bark of ash hairly root, but it can not produce gentiopicrin.In addition, Qi Xiangjun etc. use medicine source plant resource bark of ash cells produce gentiopicrin, and its gentiopicrin output is only 0.242mg/g, and its fermentation period is long.Above-mentioned research all fails to obtain desirable result at present.Aspect bark of ash development of resources research, abroad not yet report.Therefore, need badly and seek new Research Thinking solution gentiopicrin source shortage problem.
In recent years, separating natural product biosynthesis gene, and by yeast cell factory in a large number and economically biosynthesizing natural plant product become one of effective way.Yet plant terpene compound, as the senior terpenes such as monoterpene, sesquiterpene and diterpene not only have special route of synthesis, and has unique enzyme reaction mechanism.Meanwhile, many known pathways metabolisms can not be as a reference, and Secondary Metabolism of Plant network is many and complicated, adds that the crucial metabolic enzyme gene of part, in low expression level, is difficult to obtain and express from protein molecular level.
In recent years, before natural plant product biosynthesis gene information and biosynthetic pathway are not yet illustrated, L ü etc. inject mediation Ephedra genome DNA transformation yeast by Ar+ and N+, obtain inheritance stability Yeast engineering bacteria, and ephedrine and pseudoephedrine output are respectively 18.85mg/L and 4.11mg/L.Jin etc. utilize low energy ion to inject Mediated Glycyrrhiza uralensis genomic dna transformed yeast, have obtained the Yeast engineering bacteria of inheritance stability, and the production peak of its pentacyclic triterpene glycoside material Potenlini reaches 114.49mg/L.Meanwhile, because yeast self exists mevalonic acid (Mevalonate, i.e. MVA) approach, can oneself synthesize terpene substances precursor Isoprenoid, this is for utilizing the synthetic terpenoid gentiopicrin of microorganism that a good basic platform is provided.At present, abroad there is not yet and utilize low energy ion to inject the research report that mediation medicinal plant genomic dna transformed yeast technique construction produces the recombinant bacterium of effective medicinal components.Specifically, aspect the synthetic gentiopicrin of genetic modification yeast bio, there is not yet report both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in gentiopicrin biosynthesizing thereof.
For achieving the above object, the present invention has adopted following technical scheme.
A multiple-shaped nuohan inferior yeast recombinant bacterial strain, the Classification And Nomenclature of this recombinant bacterial strain is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCC No.8998.
The screening method of described recombinant bacterial strain, comprises the following steps:
1) use low energy ion to inject mediation bark of ash genomic dna and transform multiple-shaped nuohan inferior yeast starting strain;
2) recombinant bacterial strain primary dcreening operation: picking in YPD solid medium, cultivate through step 1) bacterium colony of starting strain after processing carries out YPD liquid fermenting, centrifuging and taking fermented liquid, adopt Molish reaction and Fehling test to analyze the fermented liquid of recombinant bacterial strain, in preliminary qualitative analysis fermented liquid, whether have gentiopicrin;
3) recombinant bacterial strain obtaining through qualitative method primary dcreening operation is further carried out to YPD liquid fermentation and culture, utilize thin-layer chromatography and efficient liquid phase chromatographic analysis fermentation gained fermented liquid, thereby to the further Screening and Identification of recombinant bacterial strain.
Described starting strain is multiple-shaped nuohan inferior yeast H.polymorpha DL-1 (source is ATCCNo.26012).
The condition that described low energy ion injects is: injection ion is N+, and implantation dosage is 1.5 * 10
16～2.5 * 10
16ions/cm
2, Implantation Energy is 15～25KeV, and the burst length is 5～10s, and be 5～10s interval time, and vacuum tightness is 1.5～2.0 * 10
-3pa.
The fermentation process of described recombinant bacterial strain comprises the following steps: in 37 ℃, rotating speed, be to cultivate 60～96h under 300r/min condition.
The substratum that described fermentation adopts is YPD liquid nutrient medium, and in fermenting process, stream adds 0.5% D/W, and flow velocity is 12.5mL/h.
The application of above-mentioned multiple-shaped nuohan inferior yeast recombinant bacterial strain (CGMCC No.8998) in gentiopicrin biosynthesizing.
Beneficial effect of the present invention is embodied in:
The present invention transforms in yeast starting strain at random by low energy ion implantttion technique mediation bark of ash genomic dna, obtain a strain multiple-shaped nuohan inferior yeast recombinant bacterial strain, this recombinant bacterial strain is biosynthesizing gentiopicrin effectively, have advantages of be easy to height density fermentation, product is easy to separation and Extraction, for the suitability for industrialized production of gentiopicrin provides new way, for solving gentiopicrin medicine source shortage problem, provide New methods in working.
Accompanying drawing explanation
Fig. 1 is that tunning TLC analyzes collection of illustrative plates, wherein, 1: gentiopicrin standard substance, 2: recombinant bacterial strain fermentation broth sample, 3: negative control yeast strain (low energy ion is used TE damping fluid incubation, the wash-out that does not contain bark of ash genomic dna after injecting) fermentation broth sample.
Fig. 2 is fermentation production HPLC collection of illustrative plates, wherein, and A: gentiopicrin standard substance, B: recombinant bacterial strain fermentation broth sample, C: negative control yeast strain fermentation broth sample.
Fig. 3 is the fermentograph of recombinant bacterial strain.
Fig. 4 is the genetic stability collection of illustrative plates of recombinant bacterial strain.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
The present invention be take H.polymorpha DL-1 as starting strain (source is ATCC No.26012), utilize low energy ion irradiation to inject the random transformed yeast of mediation bark of ash genomic dna, by qualitative, quantitative screening, obtain the restructuring yeast strains that produces gentiopicrin, and it is carried out to genetic stability and corresponding fermentation test.
(1) substratum
Consisting of of YPD liquid nutrient medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, pH is 6.5;
Consisting of of YPD solid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, agar 20g/L, pH is 6.5.
(2) method of screening institute foundation
(1) iridoid glycosides material detects
Molish reaction: get 800mL fermented liquid, concentrated broth, to 5mL (obtaining concentrated broth), adds 10mL methanol extraction at 100 ℃, filters extraction liquid.Get filtrate 3mL, add two Molish reagent (10% naphthyl alcohol ethanolic soln), carefully add the about 1mL vitriol oil (18.4mol/L), be sure not to shake up, observe the colour-change of two-layer liquid level intersection, occur that purple ring is positive.
Fehling test: get above-mentioned concentrated broth 3mL and join in 3mL Fehling reagent (be comprised of first and the second grade volume, first is the aqueous sodium hydroxide solution of 0.1g/mL, and second is the copper sulfate solution of 0.05g/mL).After shaking up, put in boiling water bath and boil 5min, take out coolingly, observe precipitation and colour-change, occur the positive of brick-red precipitation.
(2) thin-layer chromatography (TLC) qualitative detection: the point sample on GF254 silica gel thin-layer plate by the above-mentioned concentrated broth of 5 μ L and gentiopicrin standard substance respectively, at developping agent chloroform-methanol (V/V, 20:80), launch, after end, under ultraviolet lamp, observe, record spot, and calculate its Rf value.
(3) HPLC detection by quantitative: get 800mL fermented liquid, at 100 ℃, be concentrated into after 5mL (obtaining concentrated broth), add 10mL methanol extraction, filter extraction liquid, filtrate is measured the content of gentiopicrin for HPLC method after with 0.45 μ m filtering with microporous membrane; HPLC chromatographic condition is: chromatographic column: chemical bond mould assembly octadecyl post (SciC18 chromatographic column); Pump:K-1001; Detecter:K-1501; Moving phase: methanol-water (V/V, 30:70); Column temperature: 25 ℃.
(3) restructuring and screening process
1) extract bark of ash genomic dna: get the fresh blade 5g of road, appropriate Shaanxi real estate bark of ash, with liquid nitrogen, be ground to rapidly after powder, packed in the centrifuge tube of 50mL; Then rapidly by the CTAB extracting solution of 3mL preheating (2%CTAB, 1.4M NaCl, 0.02M EDTA, 0.1M Tris-HCl, 0.2% mercaptoethanol, pH=8.0) is added in described centrifuge tube; Put into afterwards 65 ℃ of water-baths, after insulation 60min (during jog 5 times), add 4mL chloroform-primary isoamyl alcohol (24:1), jog mixes to oyster white, is placed in 4 ℃, the centrifugal 15min of 11000rpm; Get supernatant liquor and add the Virahol of 0.6 times of volume, shake up; Be placed on 4 ℃, the centrifugal 10min of 11000rpm, abandon supernatant, 70% ethanol rinsing 2 times for precipitation, is positioned over and in Bechtop, dries up to obtain bark of ash genomic dna.With 1mL Tris-EDTA (pH8.0) damping fluid, dissolve bark of ash genomic dna, and survey its concentration with ultraviolet spectrophotometer, finally put into 4 ℃ of Refrigerator stores.
2) the ion implantation mycoderm of low energy N+: by the starting strain of this laboratory preservation (H.polymorphaDL-1) on 37 ℃, YPD solid medium after line activation 24h, the fresh colony inoculation of picking is in YPD liquid nutrient medium, under 37 ℃, 110r/min speed conditions, cultivate 16h and obtain bacterium liquid A, getting appropriate bacterium liquid A, with sterilized water, to be diluted to concentration be 1.0 * 10
7cFU/mL obtains mycelium dilution liquid, get 0.1mL mycelium dilution liquid and evenly coat the aseptic plate central authorities of diameter 90mm, sterile wind dries up makes mycoderm, and the vacuum target chamber that is then placed in ion beam implanter carries out low energy N+ implantation, Implantation Energy is 20Kev, and implantation dosage is 2.0 * 10
16ions/cm
2, the burst length is 5s, and be 10s interval time, and vacuum tightness is 1.5 * 10
-3pa.
3) solid culture: through step 2), the Tris-EDTA damping fluid (pH8.0) that contains bark of ash genomic dna with 2mL soaks the mycoderm after low energy N+ is ion implantation, after 37 ℃ of incubation 2h, with pipettor, carry out wash-out, bacterium is all eluted to obtain to bacterium liquid B, get respectively after 0.1mL bacterium liquid B evenly coats YPD solid medium and cultivate 72h in 37 ℃.
4) recombinant bacterial strain primary dcreening operation: through step 3), the bacterium colony of growing on YPD solid medium is transferred to and in test tube, carries out liquid culture (37 ℃, 110rpm/min cultivate 96h), in the centrifugal 10min of 10000r/min, collect fermented liquid and concentrate, and then detect analysis.
Gentiopicrin belongs to iridoid glycosides, and the detection of such material mainly adopts Molish reaction and Fehling test.First utilize Molish reaction to analyze restructuring bacterial strain fermentation liquor, in the reaction of observation sample liquid, whether occur purple ring (positive findings is for occurring purple ring).Then, whether recycling Fehling test occurs that to above-mentioned the fermented liquid of the recombinant bacterium of purple ring further analyzes, observe in sample liquid and have brick-red precipitation to generate (positive findings is for occurring brick-red precipitation).Above-mentioned two kinds of positive reaction results show to have glucoside compound in the fermented liquid of recombinant bacterial strain, can be used as the primary dcreening operation recombinant bacterial strain of subsequent analysis.
5) qualitative, detection by quantitative: primary dcreening operation recombinant bacterial strain is inoculated in the test tube containing 10mL YPD liquid nutrient medium, and in 37 ℃, under 110r/min, cultivate 12h and obtain seed culture fluid, the inoculum size that the volume of take is 5% is inoculated in seed culture fluid in the 250mL triangular flask containing 100mL YPD liquid nutrient medium, then in 110r/min, at 37 ℃, cultivate 90h, then in the centrifugal 10min of 10000r/min, after rinsing twice with sterile distilled water, the centrifugal thalline obtaining dries to the constant weight analysis of weighing in 85 ℃, the centrifugal supernatant liquor obtaining is qualitative for product, detection by quantitative, main thin-layer chromatography (TLC) and the high performance liquid chromatography (HPLC) of adopting.
TLC Qualitative Identification: with kapillary, concentrated broth and the gentiopicrin standard substance of the primary dcreening operation recombinant bacterial strain concentrated broth of 5 μ L, negative control yeast strain are put on thin layer plate respectively, then thin layer plate being placed in to developping agent chloroform-methanol (V/V, 20:80) launches.Result as shown in Figure 1, on the position corresponding with gentiopicrin standard substance, there is same spot in primary dcreening operation recombinant bacterial strain concentrated broth, its Rf value is 0.3, very approaching with gentiopicrin standard substance Rf value (0.29), and spot does not appear in the concentrated broth of negative control yeast strain at this place, show that primary dcreening operation recombinant bacterial strain fermenting process has novel substance synthetic.
HPLC standard measure is identified: respectively the concentrated broth of primary dcreening operation recombinant bacterial strain concentrated broth, negative control yeast strain and gentiopicrin standard substance are carried out to HPLC analysis, result as shown in Figure 2, retention time is in 12.377min place primary dcreening operation recombinant bacterial strain concentrated broth, to go out peak (Fig. 2 B), the retention time 12.370 that goes out peak (Fig. 2 A) with gentiopicrin standard substance is basically identical, and the concentrated broth of negative control yeast strain does not go out peak at this place, show that primary dcreening operation recombinant bacterial strain has the ability of biosynthesizing gentiopicrin.Pass through above-mentioned steps, final the present invention's screening has obtained a strain recombinant bacterial strain DL-49, the Classification And Nomenclature that the present invention screens the recombinant bacterial strain of acquisition is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), this recombinant bacterial strain has been preserved in (address:, preservation date is on April 3rd, 2014, China Committee for Culture Collection of Microorganisms's common micro-organisms center No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica); Deposit number is CGMCC No.8998.
(4) fermentation of recombinant bacterial strain and gentiopicrin separation and Extraction
The zymotechnique of recombinant bacterial strain (CGMCC No.8998): adopt 15L automatic fermenter to carry out high density fermentation to the recombinant bacterial strain screening (CGMCC No.8998) and produce gentiopicrin, by recombinant bacterial strain, after seed culture, (with reference to the acquisition of seed culture fluid above) is inoculated in YPD liquid nutrient medium with the inoculum size of 5% (volume ratio), in 37 ℃, rotating speed is the 96h that continuously ferments under 300r/min, pH is 6.5, in fermentation, stream adds 0.5% (w/v simultaneously, in every 100mL water containing 0.5g glucose) D/W, flow velocity is 12.5mL/h, after fermentation 48h, every 12h, get fermented liquid 500mL, measure yeast cell biomass and gentiopicrin.Result as shown in Figure 3.Known, cell concentration is along with the increase of incubation time constantly raises, and after cultivation 72h, cellular biomass is increased to 42.35g/L; Gentiopicrin starts a large amount of appearance from 60h, and along with time lengthening, output slightly increases, and during 72h, output reaches the highest, reaches 7.53mg/L.
Gentiopicrin separation and purification: according to above-mentioned zymotechnique, recombinant bacterial strain (CGMCC No.8998) is carried out to high density fermentation 96h, get respectively 500mL fermented liquid in 48h, 60h, 72h, 84h and 96h and carry out separation and purification detection analysis.Be specially fermented liquid 500mL after the centrifugal 10min of 12000r/min, supernatant liquor is concentrated into 10mL in 80 ℃, adopt D301 type macroporous resin to carry out Static Adsorption enrichment 8h to gentiopicrin, with the aqueous ethanolic solution of 20mL volume fraction 8%, gentiopicrin is carried out to wash-out afterwards, collecting elutriant carries out HPLC after being concentrated into 1mL and detects and analyze in 60 ℃, can obtain purity and be 84.35% product, the rate of recovery of gentiopicrin is 73.15%.
(5) genetic stability of recombinant bacterial strain
Will recombinant bacterial strain (CGMCC No.8998) be inoculated in the triangular flask containing 200mL YPD liquid nutrient medium after activation and cultivate (37 ℃, 110r/min), go down to posterity and cultivated for 8 generations and measure the genetic stability of recombinant bacterial strain.Result as shown in Figure 4, the gentiopicroside in different morphological that shows to go down to posterity in culturing process in recombinant bacterial strain fermented liquid is substantially constant, going down to posterity, to cultivate gentiopicroside in different morphological after 8 generations be 2.106mg/L, gentiopicroside in different morphological (2.235mg/L) than first-generation recombinant bacterial strain has reduced by 6.1%, and this recombinant bacterial strain inheritance stability is described.
In a word, the present invention utilizes low energy ion implantttion technique transformed yeast build to produce gentiopicrin recombinant bacterial strain, and screening has obtained the recombinant bacterial strain (CGMCC No.8998) that a strain can be used for fermentative production gentiopicrin.In addition; compare with plant extraction method; the synthetic gentiopicrin of microorganism fermenting organism have production cost low, with short production cycle, be not subject to natural climate to affect, saves the advantages such as soil, extracellular products separation and Extraction are simple, this for solving gentiopicrin source shortage problem, protect gentianaceae plant resource and realize its Sustainable development new way is provided.The present invention adopts the total DNA of low energy ion implantttion technique mediation bark of ash random conversion with screening in yeast to produce gentiopicrin recombinant bacterial strain first, for utilizing microorganisms producing gentiopicrin that practical basis is provided.
Claims (4)
1. a multiple-shaped nuohan inferior yeast recombinant bacterial strain, is characterized in that: the Classification And Nomenclature of this recombinant bacterial strain is multiple-shaped nuohan inferior yeast (Hansenula polymorpha), and described recombinant bacterial strain is preserved in CGMCC, and deposit number is CGMCC No.8998.
2. a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain according to claim 1, is characterized in that: the fermentation process of described recombinant bacterial strain comprises the following steps: in 37 ℃, rotating speed, be to cultivate 60～96h under 300r/min condition.
3. a kind of multiple-shaped nuohan inferior yeast recombinant bacterial strain according to claim 2, is characterized in that: the substratum that described fermentation adopts is YPD liquid nutrient medium, and in fermenting process, stream adds 0.5% D/W, and flow velocity is 12.5mL/h.
4. the application of multiple-shaped nuohan inferior yeast recombinant bacterial strain in gentiopicrin biosynthesizing as claimed in claim 1.
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| CN104593507A (en) * | 2015-01-23 | 2015-05-06 | 陕西科技大学 | Method for rapidly screening recombinant yeast capable of producing gentiopicroside based on multiplex PCR |
| CN104630355A (en) * | 2015-01-23 | 2015-05-20 | 陕西科技大学 | Method for quickly screening recombinant yeast strains producing gentiopicroside |
| CN104593507B (en) * | 2015-01-23 | 2016-08-24 | 陕西科技大学 | A kind of multiplex PCR rapid screening produces the method for gentiopicrin recombination yeast |
| CN104630355B (en) * | 2015-01-23 | 2016-08-24 | 陕西科技大学 | A kind of rapid screening produces the method for gentiopicrin restructuring yeast strains |
| CN107189953A (en) * | 2017-05-19 | 2017-09-22 | 宁波市疾病预防控制中心 | Multiple-shaped nuohan inferior yeast recombinant bacterial strain and its application in glycerine biosynthesis |
| CN107189953B (en) * | 2017-05-19 | 2019-08-27 | 宁波市疾病预防控制中心 | Multiple-shaped nuohan inferior yeast recombinant bacterial strain and its application in glycerol biosynthesis |
| CN108893494A (en) * | 2018-05-17 | 2018-11-27 | 陕西科技大学 | Betulic acid biosynthesis pathway screening technique based on multiple-shaped nuohan inferior yeast |
| CN108893494B (en) * | 2018-05-17 | 2021-03-05 | 陕西科技大学 | Screening method of betulinic acid biosynthesis pathway based on Hansenula polymorpha |
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